1. Field of the Invention
This invention relates generally to apparatus and methods for testing the susceptibility of microorganisms and mammalian cells to growth inhibition by antimicrobial and cytotoxic products. This invention further relates to both qualitative and quantitative susceptibility testing.
Microorganism and cellular specimens to be tested may be supplied to a laboratory from a number of sources. The specimens may be collected by doctors in their offices and sent to a central testing laboratory or the specimens may be collected from patients in hospital with which the laboratory is associated. The microorganism specimens may come from various parts of the body, for example, from cerebral spinal fluid, an abscess, an infected wound, genital infections, etc. The cellular specimens will usually be from tumor biopsies specimens. The collected specimens are cultured on a suitable media in accordance with normal laboratory practice. From the bacterial colonies and cellular clones on the primary culture plate, an inoculum is prepared in accordance with an established procedure which produces a bacterial or cellular suspension of a prearranged concentration. Further processing of the suspension depends on the apparatus and method to be used for susceptibility testing.
The purpose of bacterial susceptibility testing is to provide information to the referring physician on the probable success of the antibiotic drug therapy that has already been initiated. The physician will generally prescribe an antimicrobial product, commonly called an antibiotic drug, to be administered before the test results are known, but it is often important for the physician to learn whether that antimicrobial product and/or the concentration given will successfully kill the microorganism that is causing the infection. After the test results are in, the physician can change the drug therapy if the test results show that there is a reason to do so.
The purpose of cellular cytotoxicity testing is usually to determine the susceptibility of the tumor cells to particular chemotherapeutic drugs.
The term qualitative susceptibility testing refers to testing apparatus and methods which produce test results that generally indicate whether an organism or cellular specimen is sensitive or resistant to a particular antibiotic or cytotoxic product. Depending on the method involved only one or two concentrations of antimicrobial product are usually utilized. The degree of sensitivity or resistance is not reported in qualitative susceptibility testing.
The term quantitative susceptibility testing refers to testing apparatus and methods which produce test results that provide data on the concentration of the antimicrobial or cytotoxic product that will be sufficient to inhibit growth of the microorganism or tumor cells. Typically, for microorganism specimens, six or more different dilutions of the antimicrobial product are utilized covering the therapeutic range of concentrations of the antimicrobial product. The term Minimum Inhibitory Concentration (MIC) is often used to refer to the result provided by quantitative susceptibility testing of microorganism and is defined as the minimum concentration of the antimicrobial product which will produce inhibition of the growth of the microorganism.
The term antimicrobial product will be used herein to designate a product that contains one or more antimicrobial agents (i.e. individual antibiotics) in prearranged concentrations and is thus a general designation for a single antibiotic drug or a broad spectrum formulation that contains more than one antibiotic agent. The term cytotoxic product will be used herein to designate a product that contains one or more drugs which are capable of inhibiting the growth of human and/or other mammalian tumor cells.
The use of oxidation-reduction (redox) indicators for antimicrobial susceptibility testing has been proposed but has not yet found widespread use. In theory, microbial growth can be detected by incubation with a redox indicator which changes color when reduced from an oxidized state to a reduced state by the metabolic processes of the microorganism. Particular indicators which have been proposed for such testing include tetrazolium and resazurin.
Of particular interest to the present invention is the use of resazurin as a redox indicator for detecting microbial growth in susceptibility tests. Test protocols which have been proposed include incubation of sample containing a test organism in a growth medium containing resazurin and an antimicrobial agent. Growth inhibition by the antimicrobial agent can then be detected by visually comparing the color of the growth medium with the color of the medium of a test sample containing growth medium but no antimicrobial agent.
While such test protocols utilizing resazurin promise convenience, accuracy, and use with many types of organisms, they have suffered from certain limitations which have prevented their widespread use. In particular, resazurin is subject to autoreduction in most growth media, i.e. the resazurin will be reduced and change color even when microbial growth is absent. Thus, the use of negative test controls is problematic, particularly over extended time protocols such as overnight tests. Many non-stabilized media, such as brain heart infusion broth, cause reduction of the resazurin over time and are generally unsuitable for performing protocols over periods approaching 24 hours.
Autoreduction of the resazurin is less of a problem with relatively short test protocols, i.e. several or fewer hours, but such short protocols are less useful in detecting growth of weakly growing microorganisms. Indeed, many weakly growing organisms require extended incubation protocols approaching 24 hours, which protocols are rendered difficult or impossible because of the media instability problems just discussed.
Thus, it would be particularly desirable to provide improved media and methods which employ resazurin as a microbial growth indicator, where the media and methods are particularly stable and permit extended incubation protocols without significant autoreduction.
2. Description of the Background Art
The use of absorbent pads impregnated with resazurin and antibiotics for antimicrobial susceptibility testing is described in Baker et al. (1980) Microbiol. 26:248-253 and Canadian Patent 1,112,140. Bacterial isolates are applied to the pad in a brain heart infusion broth. The protocols described, however, are generally not suitable for determining minimum inhibitory concentrations (MIC). Kanazawa et. al. (1966) J. Antibiotics 19:229-233 also describes the use of absorbent pads impregnated with resazurin and antimicrobials for susceptibility testing. Brown et al. (1961) J. Clin. Path. 5:10-13 and U.S. Pat. No. 3,107,204 describe the use of absorbent pads impregnated with another redox indicator (tetrazolium) and antimicrobials in susceptibility testing. Japanese patent publication 2-211899 teaches that resazurin susceptibility tests may run in Mueller-Hinton broth and may be detected based on fluorescence. Japanese patent publications 2-12056, 43-19817, and 42-25409 are also relevant to susceptibility assays employing resazurin as a redox indicator.
The full disclosures of each of these references are incorporated herein by reference.